The present invention relates to chemical control of gene expression in plants. In particular, it relates to a method whereby juvenile hormone or one of its agonists is used as a chemical ligand to regulate receptor-mediated expression of a target polypeptide in a plant cell, as well as to transgenic plant cells, plant material or plants and the progeny thereof containing appropriate expression cassettes.
In some cases it is desirable to control the time or extent of expression of a phenotypic trait in plants, plant cells or plant tissue. An ideal situation would be the regulation of expression of such a trait at will, triggered by a chemical that could be easily applied to field crops, ornamental shrubs, etc. One such system of regulating gene expression which could be used to achieve this ideal situation, as yet unknown to be present naturally in plants, is the steroid and thyroid hormone superfamily of nuclear receptors. The steroid and thyroid hormone superfamily of nuclear receptors is found in mammals and insects and is composed of over 100 known proteins. Some of the receptors within this superfamily which are found in mammals are Retinoic Acid Receptor (RAR), Vitamin D Receptor (VDR), Thyroid Hormone Receptor (T3R) and Retinoic X Receptor (RXR). These and other receptors of the superfamily bind to the 5xe2x80x2 regulatory region of the target gene and, upon binding of a chemical ligand to the receptor, transactivate target gene expression.
In addition to the receptors found in mammals as described above, receptors of similar structure and activity have been indentified in the insect Drosophila. Koelle et al., Cell 67: 59 (1991); Christianson and Kafatos, Biochem. Biophys. Res. Comm. 193:1318 (1993); Henrich et al., Nucleic Acids Res. 18: 4143 (1990). The Ecdysone Receptor (EcR) binds the insect steroid hormone 20-hydroxyecdysone and, when heterodimerized with the product of the Ultraspiracle gene (USP), transactivates gene expression. Additional chemical ligands besides 20-hydroxyecdysone, such as hormone agonists, will also bind to EcR under similar conditions and cause transactivation of a target gene.
USP has also been shown to be a member of the steroid and thyroid superfamily of nuclear receptors although it is considered an xe2x80x9corphanxe2x80x9d receptor since its ligand has not been identified (Seagraves, Cell 67:225-228 (1991)). USP is related in sequence to RXRxcex1 (Oro et al, Nature, 347:298-301 (1990)), and RXR is capable of forming heterodimers with EcR (Thomas et al., Nature 362: 471-475 (1993)). Methoprene and its derivative methoprene acid, which are juvenile hormone agonists, has been shown to transcriptionally activate a recombinant reporter gene in both insect and mammalian cells by acting through RXRxcex1 (Harmon et al., Proc. Natl. Acad. Sci. 92:6157-6160 (1995)). Juvenile hormone, however, does not induce RXRxcex1-mediated transactivation (Harmon et al.) To date, there has been no definitive evidence for a nuclear juvenile hormone receptor (Harmon et al.; Henrich and Brown, Insect Biochem. Molec. Biol. 25:881-897 (1995)), although it has been suggested that the orphan receptor USP may be a candidate (Harmon et al.; see also Seagraves; Oro et al., Current Opinion in Genetics and Development2:269-274 (1992)).
Juvenile hormone and its agonists offer previously unrecognized opportunities for chemical control of gene expression in plants since these chemicals are already known for agricultural use. What has been lacking to date is a means by which these chemicals can be used to induce transactivation of a target gene in a transgenic plant. Juvenile hormone and its agonists have herein shown to be ligands for the orphan receptor USP. This discovery permits the implementation of gene control strategies for plants which utilizes a nuclear receptor that does not occur naturally in plants. This means that the only effect of the application of juvenile hormone or one of its agonists will be to induce expression of a genetically engineered target gene. As is demonstrated by the present invention, USP receptor polypeptides, and the plant-expressible genes that encode them, have been developed which function in plant cells to control expression of a target polypeptide wherein the USP receptor polypeptide activates the 5xe2x80x2 regulatory region of a target expression cassette in the presence of juvenile hormone or one of its agonists. Such a method of controlling gene expression in plants is useful for controlling various traits of agronomic importance, such as plant fertility.
The present invention is drawn to a method of controlling gene expression in plants. Specifically, the method comprises transforming a plant with a USP receptor expression cassette which encodes a USP receptor polypeptide, with at least one target expression cassette which encodes a target polypeptide, and optionally with a secondary receptor expression cassette encoding a secondary receptor polypeptide distinct from the USP receptor polypeptide. Contacting said transformed plant with juvenile hormone or one of its agonists activates or inhibits expression of the target polypeptide in the presence of said USP receptor polypeptide. Optionally, additional xe2x80x9csecondaryxe2x80x9d receptor expression cassettes may be used, wherein the secondary receptor expression cassette encodes a receptor polypeptide distinct from USP. The method is useful for controlling various traits of agronomic importance, such as plant fertility.
The invention is further drawn to transgenic plants comprising a USP receptor expression cassette and a target expression cassette capable of activation by juvenile hormone or one of its agonists. Also encompassed by the invention is a method of identifying previously unknown ligands for USP which are effective in a plant cell environment. Substances to be tested are identified by placing them in contact with plant cells transformed with a USP receptor expression cassette and a target expression cassette. The target expression cassette encodes a reporter polypeptide whose expression can be determined quantitatively or qualitatively, whereby the test substance is identified as a ligand for USP.